Junctional types in the tissues of an onychophoran: the apparent lack of gap and tight junctions in Peripatus

The Onychophora are a rare group of primitive invertebrates, relatively little investigated. Tissues from a range of their digestive, secretory and excretory organs have been examined to establish the features of their intercellular junctions. Glutaraldehyde-fixed cells from the midgut and rectum, as well as the renal organ, mucous gland, salivary gland, epidermis, CNS and testis from specimens of Peripatus acacioi, have been studied by thin section and freeze-fracture electron microscopy. Adjacent cells in the epithelia of all these tissues are joined by apical zonulae adhaerentes, associated with a thick band of cytoskeletal fibrils. These are followed by regular intercellular junctional clefts, which, in thin sections, have the dense, relatively unstriated, appearance of smooth septate junctions (SSJ). However, freeze-fracture reveals that only the midgut has what appear to be characteristic SSJs with parallel alignments of closely-packed rows of intramembranous particles (IMPs); these IMPs are much lower in profile than is common in such junctions elsewhere. The mucous gland, testis, rectal and renal tissues exhibit, after freeze-fracture, the characteristic features of pleated septate junctions (PSJ) with undulating rows of aligned but separated junctional particles. Suggestions of tricellular septate junctions are found in replicas at the interfaces between 3 cells. In addition, renal tissues exhibit scalariform junctions in the basal regions of their cells. Between these basal scalariform and apical septate junctions, other junctions with reduced intercellular clefts are observed in these renal tissues as well as the rectum, but these appear not to be gap junctions. Such have not been unequivocally observed in any of the tissues studied from this primitive organism; the same is true of tight junctions.     

Intercellular junctions in myriapods

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.     

The Organization of Actin in the Apical Region of Insect Midgut Cells after Deep Etching

The midgut ofTenebriolarvae, which reveals a strong reaction for F-actin beneath the apical microvilli after rhodamine—phalloidin treatment, was studied to examine localization of actin. Freeze-fracture replicas of the lateral midgut borders reveal that smooth septate junctions with their characteristic rows of aligned intramembranous particles (IMPs) are found on the upper third of these borders. Thin sections show that short punctate adhering junctions may also occur on this part of the border. Deep etching reveals that the rows of septate junctional IMPs are closely juxtaposed to cytoplasmic fibrils that demonstrate the structural features typical of actin as well as heavy meromyosin labeling. These actin fibrils appear to insert into the junctional membranes. Hence cytoskeletal elements have an intimate spatial association with the membrane modifications typical of intercellular septate junctions and may be involved in the positioning of their component IMPs and also possibly of their septal ribbons.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Intercellular junctions in the hepatopancreas of the lobster Nephrops norvegicus

The hepatopancreas of the lobster has recently been found to be a rich source of material from which to isolate arthopod gap junctions biochemically (Finbow et al., 1983a; 1984). It has therefore been studied here to assess the features of these intercellular junctions and any others that may be present, in vivo. The tissue consists of columnar epithelial cells which possess apical microvilli and basal infoldings. In thin sections the lateral borders of these cells are characterized by desmosomes and smooth septate junctions as well as by gap junctions. The desmosomes exhibit no apparent freeze fracture profile but the septate junctions display parallel rows of ridges or aligned intramembranous particles (IMPs) with complementary grooves on the other membrane half; these IMPs shift in their preferential fracturing plane depending on whether the tissue has first been fixed, always remaining on the EF if unfixed. The IMPs or connexons, of which the gap junctions are composed, fracture onto the E face, leaving complementary pits on the P face, regardless of whether the tissue is fixed or not. At the base of the pancreatic cells, the lateral borders are thrown into interdigitating folds which display endocytotic profiles and possible internalization of junction-bearing membranes. This phenomenon, which is readily visualized both after tracer incubation and in replicas, may represent junctional degradation relating to membrane turnover.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.     

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta)

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deepetching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.     

Fine structure of the rectum in cockroaches (Dictyoptera): General organization and intercellular junctions

The organization of the rectal pads is described in cockroaches belonging to the Groups Blattoidea (Periplaneta americana, Blatta orientalis) and Blaberoidea (Supella supellectilium, Blaberus craniifer). In the Blattoidea, each pad is composed of two layers (principal and basal cells) and is surrounded by very narrow junctional cells supporting the sclerotized cuticle of the pad frame; basally, the junctional cells abut on to the basal cells. In the Blaberoidea, the basal cell layer is discontinuous, the basal cells being interspersed between extensions of the junctional cells beneath the pad. The ultrastructural features of each cell type is described, with special reference to the intercellular junctions, which exhibit unusual complexity. Four types of junction are recognized: desmosomes (belt and spot desmosomes), gap junctions, septate junctions and scalariform (ladder-like) junctions. The last are usually closely associated with mitochondria, forming mitochondrial-scalariform junction complexes (MS). The distribution of these junctions is examined in relation to the partitioning of extracellular spaces, and to the problem of fluid transport.     

Fine structure of the rectal pads in the desert locust Schistocerca gregaria with reference to the mechanism of water uptake

The rectal pads of Schistocerca gregaria are composed of three different cell types: epithelial, secondary and junctional cells. The rectal pads are interconnected by simple rectal cells and both are lined internally by a articular intima. The epithelial cells exhibit extensive infoldings of the apical plasma membranes that are closely associated with mitochondria. Their lateral plasma membranes are highly folded around large mitochondria and enclose intercellular channels and spaces. They are united by belt and spot desmosomes, septate junctions, gap junctions and scalariform junctions, but terminate in a basal syncytium without contacting the basal plasma membranes. The apical and basal cytoplasm contain coated vesicles, dense tubular elements, multivesicular bodies and lysosomes, suggesting receptor-mediated endocytosis of small peptide molecules into the epithelial cells. The apical membrane infoldings of the secondary cells are also associated with large mitochondria. Their basal plasma membranes are covered by connective cell processes and connected with them by spot desmosomes which may be involved in solute recycling. The presence of neurosecretory-like axons near the secondary cells suggests that they exert local control on the function of these cells. The ultrastructural details are examined in relation to their role in solute and water transport.     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Intercellular junctions in the corpora cardiaca of locusts

Summary The intercellular junctions in the corpora cardiaca of the locusts Schistocerca gregaria and Locusta migratoria were investigated by transmission electron microscopy. In the glandular lobes, complexes consisting of scalariform junctions and associated mitochondria, comparable to those previously observed in ion transporting epithelia, are formed between gland cells, and more rarely between gland cells and the neurons innervating them. Their structure and abundance are apparently unaffected by the stage of development or by the various experimental conditions employed. In the neural lobe, scalariform junctions form between glial cells and show close association with the endoplasmic reticulum. Gap junctions are present among glandular, neural and glial elements, and are formed between cells of the same type and of different types. Contacts resembling punctate tight junctions are widely distributed in the gland, but would be unlikely to form a barrier to diffusion. Septate junctions are formed exclusively between glial cells.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.     

Structural diversity of gap junctions. A review.

Gap junctions are plasma membrane specializations characterized as aggregates of intramembranous particles in two apposed membranes meeting particle-to-particle in the 2-4 nm intermembrane 'gap'. Recent thin-section and freeze-fracture evidence has revealed significant structural variations of gap junctional structure at various stages of development and from different organisms and tissues. It is suggested that a comparative analysis of these differences may provide clues to the specific biological functions(s) of these ubiquitous organelles.     

Cell junctions in the gut of Protura

The main cell junctions in the intestinal tract of a small group of apterygotan insects, Protura, were examined in conventional thin sections, tracer-infiltrated sections and freeze-fracture replicas. The smooth septate junctions in the midgut of collembolan Tomocerus minor were also studied for comparison. Pleated septate junctions are found in foregut, hindgut and Malpighian papillae. They exhibit regular septa crossing the intercellular clefts in thin sections; and the septa with a pronounced zig-zag appearance run parallel to form a honeycomb structure in tracer-impregnated sections. After freeze-fracture undulating rows of intramembranous particles (IMPs) are visible on the P face. Smooth septate juncions are observed in the midgut. The interceullar septa often run in pairs for long tracts and exhibit a wavy course in lanthanum impregnated sections. The IMPs exhibited on the E face are usually separated one from another. Twin arrangement of particle rows is also apparent on the replicas. Gap junctions are frequent in both the midgut and hindgut and possess the conventional characteristics of 'inverted gap junction' with E face connexons. These results provide further evidence relating Protura closely to Collembola as well as to primitive arthropods.     

A freeze-etch study of the central myelin

The intramembranous organization of the interparanodal myelin lamellae in CNS was investigated by means of the freeze-etching technique. The density of the IMPs is about equal on both P- and E- fracture faces of every myelin sheath, but varies between the myelin sheaths. The morphology of the myelin tight junctions is also described. The nature of the IMPs and the functional significance of their mode of distribution are discussed.     

Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia

We have used freeze-fracture electron microscopy to investigate the relationship between the formation of the tight junction in the establishment of a differential distribution of intramembranous particles (IMPs) between the luminal and basolateral membranes of a canine kidney cell line (MDCK). This involves a characterization of the IMP distribution in these membranes in confluent monolayers of MDCK cells, in EGTA-dissociated cells, and in cells at various stages of reassociation. While normal confluent MDCK monolayer cultures exhibit tight junctions and an IMP differential distribution between the luminal and basolateral membranes, cultures dissociated with EGTA lose both formed tight junctional elements and the differential IMP distribution. We have also found that as tight junctions reform between reaggregating MDCK cells, intramembranous particles appear to rapidly redistribute with respect to them. An asymmetric distribution of these particles in the luminal and basolateral membranes is eventually achieved. Tight junction formation appears so closely linked to the genesis of IMP polarity that at early time points when only a string of tight junctional components spans the junctional zone, differential IMP distributions are seen. Thus, our dissociation studies suggest a close relationship between the integrity of the tight junction and the maintenance of IMP polarity between the luminal and basolateral membranes, while cell reassociation studies suggest that the tight junction may be functionally linked to the genesis of IMP polarity.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

Pronase reduces intramembranous particle density in uterine epithelial cells in vivo

The proteolytic enzyme, pronase, was injected into the uterine lumen of rats. This treatment removed half the intramembranous particles (IMPs) from the apical plasma membrane of the uterine epithelial cells but tight junctions of these cells were unaffected. We conclude that at least some of the IMPs are proteinaceous in nature and suggest that IMPs not affected by pronase may be deeply embedded in the lipid bilayer.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.